Functional characterisation of microRNA-containing Argonaute protein complexes

microRNAs (miRNAs) are small non-coding RNAs of 21-24 nt in size,
which are endogenously expressed in higher eukaryotes and play
important roles in processes such as tissue development and stress
response and in several diseases including cancers. In mammals,
miRNAs guide proteins of the Argonaute family (Ago proteins) to
partially complementary sequences typically located in the
3’-untranslated regions (3’-UTRs) of specific target mRNAs, leading
to translational repression or mRNA degradation. To gain further
insight into the function of human miRNAs, we analyzed the protein
as well as the RNA composition of miRNA-Ago protein complexes in
molecular detail. To identify novel Ago-interacting proteins, we
isolated Ago complexes and investigated them by mass spectrometry
and co-immunoprecipitation experiments. We found that trinucleotide
repeat-containing 6B (TNRC6B), Moloney leukemia virus 10 (MOV10),
RNA binding motif protein 4 (RBM4) and Importin 8 (Imp8) interact
with human Ago proteins. Moreover, using RNA interference and EGFP
and dual luciferase reporter assays, we demonstrated that these
factors are required for miRNA function, indicating that we have
identified new components of the miRNA pathway. Intriguingly,
depletion of Imp8 does not affect the levels of mature miRNAs or
the interaction of miRNAs with Ago proteins, but is required for
efficient association of Ago-miRNA complexes with their target
mRNAs. Thus, Imp8 is the first factor acting at the level of target
mRNA binding, establishing a novel layer of regulation for the
miRNA pathway. Imp8 is an Importin-β-like protein, which has
previously been implicated in nuclear import of substrate proteins.
In line with these results, we demonstrated that a detectable
fraction of Ago2 localizes to the nucleus of human cells. Moreover,
knockdown of Imp8 by RNAi reduces the nuclear signal of Ago2,
suggesting that Imp8 affects the nuclear localization of Ago2.
Therefore, our data suggest that Imp8 has a dual function both in
the cytoplasmic miRNA pathway and in nuclear transport of Ago
proteins. To identify small RNAs, which associate with human Ago
proteins, we isolated, cloned and sequenced small RNAs bound to
Ago1 and Ago2 complexes. In addition to known miRNAs, we found
several small RNAs, which derive from small nucleolar RNAs
(snoRNAs). We therefore investigated the function of one particular
small RNA, which is derived from the snoRNA ACA45 and showed that
it functions like a miRNA. Interestingly, this small RNA is
processed by the miRNA maturation factor Dicer, but does not
require the microprocessor complex that is essential for processing
of primary miRNA transcripts. Thus, we have identified a novel
biogenesis pathway of a new class of small RNAs that can function
like miRNAs. To experimentally identify mRNAs that are stably
associated with miRNA-Ago protein complexes, we isolated and
analyzed Ago1 and Ago2-bound mRNAs by cloning and sequencing and by
microarray hybridization techniques. Using dual luciferase reporter
assays, we demonstrated that many Ago-associated mRNAs are indeed
miRNA targets. Therefore, we have developed a method allowing for
the identification of miRNA target mRNAs from cell lines or tissues
of interest independently of computational predictions. In a
project that was independent of our studies on Ago protein
complexes, we investigated structural and functional requirements
for the activity of small interfering RNAs (siRNAs). siRNAs are
small double-stranded RNAs of appr. 21 nt in size, which trigger
the sequence-specific endonucleolytic degradation of perfectly
complementary target transcripts upon binding to Ago2. However,
both single strands of a siRNA duplex can potentially have unwanted
“off-target effects” by repressing partially complementary target
mRNAs through binding to their 3’-UTRs. We therefore developed a
method to selectively inhibit the activity of the siRNA strand that
is dispensable for target silencing (“passenger strand”) through
chemical modification of its 5’-end. This method could be a useful
tool for the design of highly specific siRNAs. Taken together, we
have analyzed the composition of Ago-miRNA protein complexes by a
variety of methods and identified novel protein factors of the
miRNA pathway, a novel class of small RNAs as well as a panel of
previously unknown miRNA target mRNAs. The techniques for the
purification and the analysis of Ago complexes that were developed
in this study will provide useful tools for future analyses of
miRNA pathway factors, small RNAs and miRNA target mRNAs from any
tissue or cell line of interest.